Cytoplasmic polyhedrosis virus protein complex of a polyhedrin and a VP3 polypeptide

ABSTRACT

It is intended to provide a protein complex and a production process whereby the protein complex can be efficiently produced without lowering its function. It is also intended to provide use of the protein complex in a biosensor, an immobilized enzyme and so on. A protein complex comprising a polyhedral protein having an insect virus encapsulated therein and a target protein having a restricted region of a capsid protein VP3 of cytoplasmic polyhedrosis virus, more specifically, a region which is either a region from the N-terminus to the 40th amino acid residue or a region from the 41st amino acid residue to the 79th amino acid residue as an embedding signal for polyhedron, and a process for producing the same. The polyhedral protein has an effect on improvement in the stability of the target protein, protection thereof or improvement in the preservation properties thereof, or a combination thereof.

TECHNICAL FIELD

This invention relates to a protein complex, a process for producing thesame, and use of the protein complex in a biosensor, an immobilizedenzyme and so on.

BACKGROUND ART

Conventionally, a so-called protein complex, in which a protein isencapsulated in another protein, has been known. As for production ofthis type of protein complex, for example, a method of applying asolution of a dissolved protein to a surface of a crystalline protein isconsidered.

However, it is extremely difficult to carry out this method withoutdissolving the crystalline protein. Accordingly, the fact is that thismethod is hardly adopted for the purpose of protecting a useful protein(hereinafter referred to as a target protein) such as an enzyme, anantigen, an antibody, a cytokine or a receptor.

As for protection of a target protein, a method of covalently binding apolymer such as a polysaccharide polymer or polyethylene glycol to atarget protein has been adopted. This method is a method in which apolymer is bound to a functional group such as an amino group or acarbonyl group in the target protein under mild reaction conditions.However, in this method, the binding site, the catalyzed site or thelike of the target protein could not be controlled. In addition, sincethe binding site, the catalyzed site or the like varies depending on thetype of the target protein, the method could not be applied to all thetarget proteins.

As for preservation of a target protein, generally, a method ofpreservation at a lower temperature is employed. In addition, a methodof adding or mixing a protective substance (e.g., a polysaccharidepolymer, polyethylene glycol and the like), which is expected to have afunction of stabilizing the protein structure, to or with a targetprotein is also employed. However, by employing these methods, thestability or the function of the target protein was lost in some casesdue to the changes in the environment, which is an external factor. Thatis, it is because the target protein is easy to dissolve together withthe protective substance when water comes in contact, temperature orhumidity increases, or dew condensation occurs. In addition, the targetprotein is degraded or ingested together with the protective substancewhen putrefactive bacteria such as germs or fungi exist, penetrate, oremerge. Therefore, when the target protein is a polymeric protein suchas a protein molecule of some enzymes or antibodies, it lose itsfunction completely by subjecting to a change in even a part of itsstructure or by degrading a part thereof with the action of a protease.However, when the target protein is used, it is essential that itsufficiently have its function. Therefore, it is necessary to verify thestability of the target proteins in a state of preservationindividually. In the case of employing a conventional technique, it isnecessary to take the target protein out of the protective substance,therefore, not only it takes a lot of time and efforts, but also thetarget protein is susceptible to denaturation.

By the way, cytoplasmic polyhedrosis virus forms a polyhedron composedof a polyhedral protein in a cell infected with the virus during thelate phase of the viral infection, and many virus particles are embeddedin the polyhedron.

The reason why the virus particles enter specifically in this polyhedronis known and it is due to the specific relationship between a capsidprotein VP3 of the virus particle and a polyhedral protein (Non-PatentDocument 1).

In view of the above-mentioned background, the present inventorcompleted the invention, which relates to a protein complex contributingto protection, preservation and improvement in stability of a targetprotein and a process for producing the same, and applied for a patentpreviously (Patent Document 1). The object of the description of theabove-mentioned invention is to embed a polymeric target protein in thispolyhedron and to enhance the embedding efficiency. Therefore, byshortening a gene encoding a capsid protein of cytoplasmic polyhedrosisvirus, the size (molecular weight) of a protein which can be embedded ina polyhedron is made large, and this target protein is further moreefficiently embedded in the polyhedron. Further, as the method, theamino acid sequence of VP3, which is a constituent protein of theenvelope of cytoplasmic polyhedrosis virus, is introduced to theN-terminus or the C-terminus of the target protein, and this fusionprotein is expressed with a baculovirus vector. At this time, byinfecting an insect cell together with a virus expressing a polyhedralprotein of cytoplasmic polyhedrosis virus, the fusion protein isembedded in a polyhedron. Accordingly, it is necessary to fuse a cDNAencoding a constituent protein of cytoplasmic polyhedrosis virus and agene encoding a target protein so that a foreign protein expressed witha baculovirus vector, namely, a target protein is inserted at theN-terminus or the C-terminus of the constituent protein of cytoplasmicpolyhedrosis virus. At this time, it is important that the open readingframes encoding the constituent protein and the protein of the targetprotein gene are cloned in-frame. In this way, a recombinant baculovirusexpressing the constituent protein of cytoplasmic polyhedrosis virus andthe target protein as one fusion protein is constructed, which isdescribed in the above-mentioned invention.

Patent Document 1: International Patent Application WO 02/36785A1

Non-Patent Document 1: Ikeda et al., (2001) J. Virol. 75, 988-995

Applicants herein incorporate by reference the nucleic acid and aminoacid sequences of capsid protein VP3 found in FIG. 1 of Non-PatentDocument 1 (Ikeda et al., (2001) J. Virol. 75, 988-995).

DISCLOSURE OF THE INVENTION

The present invention is completed by further improving theabove-mentioned invention and identifying VP3, which is used as anembedding signal for polyhedron, within the specific area.

An object of the present invention is to provide a protein complex thatcan encapsulate a target protein whose size (molecular weight) isincreased, in addition a target protein having a fluorescent orlight-emitting function or a bioactive function, and moreover apolymeric target protein, and further can verify the function of thetarget protein in a state of a complex.

In addition, another object of the present invention is to provide aproduction process that can efficiently produce a protein complex havingany of target proteins with a variety of properties encapsulated thereinwithout lowering the function thereof.

Further, another object of the present invention is to provide use of aprotein complex in a biosensor, an immobilized enzyme and so on.

A gist of the present invention is a protein complex comprising apolyhedral protein having an insect virus encapsulated therein and atarget protein having a restricted region of a capsid protein VP3 ofcytoplasmic polyhedrosis virus as an embedding signal for polyhedron.

The restricted region of VP3 is either a region from the N-terminus tothe 40th amino acid residue or the region from the 41st amino acidresidue to the 79th amino acid residue. In this case, a gist of thepresent invention is a protein complex comprising a polyhedral proteinhaving an insect virus encapsulated therein and a target protein having,as an embedding signal for polyhedron, a restricted region, which iseither a region from the N-terminus to the 40th amino acid residue or aregion from the 41st amino acid residue to the 79th amino acid residueof a capsid protein VP3 of cytoplasmic polyhedrosis virus.

The polyhedral protein has an effect on improvement in the stability ofthe target protein, protection thereof or improvement in thepreservation property thereof, or a combination of any of these. In thiscase, a gist of the present invention is a protein complex comprising apolyhedral protein having an insect virus encapsulated therein and atarget protein having, as an embedding signal for polyhedron, arestricted region of a capsid protein of cytoplasmic polyhedrosis virus,more specifically, a restricted region, which is either a region fromthe N-terminus to the 40th amino acid residue or a region from the 41stamino acid residue to the 79th amino acid residue of VP3, in which thepolyhedral protein has an effect on improvement in the stability of thetarget protein, protection thereof or improvement in the preservationproperty thereof, or a combination of any of these.

The target protein is at least one member selected from the groupconsisting of fluorescent or light-emitting proteins, enzymes, antigens,antibodies, cytokines, receptors and bioactive proteins. In this case, agist of the present invention is a protein complex comprising apolyhedral protein having an insect virus encapsulated therein and atarget protein having, as an embedding signal for polyhedron, arestricted region of a capsid protein of cytoplasmic polyhedrosis virus,more specifically, a restricted region, which is either a region fromthe N-terminus to the 40th amino acid residue or a region from the 41stamino acid residue to the 79th amino acid residue of VP3, and being atleast one member selected from the group consisting of fluorescent orlight-emitting proteins, enzymes, antigens, antibodies, cytokines,receptors and bioactive proteins, in which the polyhedral proteinpreferably has an effect on improvement in the stability of the targetprotein, protection thereof or improvement in the preservation propertythereof, or a combination of any of these.

In addition, a gist of the present invention is a process for producinga protein complex, wherein a cell is infected with a vector that hasbeen integrated with a gene encoding a target protein together with avector that has been integrated with a gene encoding a polyhedralprotein, and the cell is cultured, whereby a protein complex having acomplex structure composed of the target protein and the polyhedralprotein is produced in the cell.

Still further, a gist of the present invention is a biosensorcharacterized in that a protein complex comprising a polyhedral proteinhaving an insect virus encapsulated therein and a target protein having,as an embedding signal for polyhedron, a restricted region of a capsidprotein of cytoplasmic polyhedrosis virus, more specifically, arestricted region, which is either a region from the N-terminus to the40th amino acid residue or a region from the 41st amino acid residue tothe 79th amino acid residue of VP3, and more specifically, being atleast one member selected from the group consisting of fluorescent orlight-emitting proteins, enzymes, antigens, antibodies, cytokines,receptors and bioactive proteins, in which the polyhedral proteinpreferably has an effect on improvement in the stability of the targetprotein, protection thereof or improvement in the preservation propertythereof, or a combination of any of these, is arranged in dots or lineson a substrate and immobilized thereon, a biosensor characterized inthat the protein complex is packed in such a manner that it can becontacted with a substance in a test solution in a recess formed on asubstrate, or a biosensor characterized in that the protein complex ispacked in a container in such a manner that it can be contacted with asubstance in a test solution.

In addition, a gist of the present invention is an immobilized enzyme inwhich a protein complex comprising a polyhedral protein having an insectvirus encapsulated therein and a target protein having, as an embeddingsignal for polyhedron, a restricted region of a capsid protein ofcytoplasmic polyhedrosis virus, more specifically, a restricted region,which is either a region from the N-terminus to the 40th amino acidresidue or a region from the 41st amino acid residue to the 79th aminoacid residue of VP3, and more specifically, being at least one memberselected from the group consisting of fluorescent or light-emittingproteins, enzymes, antigens, antibodies, cytokines, receptors andbioactive proteins, in which the polyhedral protein preferably has aneffect on improvement in the stability of the target protein, protectionthereof or improvement in the preservation property thereof, is packedin a container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a figure illustrating a method of shortening VP3 gene andpreparation of a transfer vector.

FIG. 2 is a figure showing the relationship between a shortened VP3 geneand the amino acid residues encoded by the gene.

FIG. 3 shows the determination whether or not a protein encoded by agene, in which a shortened VP3 gene has been introduced into an EGFPgene, is encapsulated in a polyhedron based on the presence or absenceof green fluorescence from the polyhedron.

FIG. 4 shows a green fluorescence intensity observed in a state whereEGFP having the nucleotide sequence of a shortened VP3 at the N-terminusis encapsulated in a polyhedron. The fluorescence intensity was gradedat five levels, 1+, 2+, 3+, 4+ and 5+.

FIG. 5 shows the results obtained by introducing the region from the39th amino acid residue to the 79th amino acid residue of VP3 into theN-terminus of Cyclin-dependent kinase 5 as a signal for encapsulation ina polyhedron, thereby encapsulating this protein in a polyhedron, andattaching the polyhedron to a slide glass and performing anantigen-antibody reaction on the surface of the polyhedron.

BEST MODE FOR CARRYING OUT THE INVENTION

A protein complex according to the present invention comprises a targetprotein having a restricted region of a capsid protein VP3 ofcytoplasmic polyhedrosis virus as an embedding signal for polyhedronencapsulated in a polyhedral protein having an insect virus encapsulatedtherein. Here, “encapsulation” means that it includes a state where atarget protein is completely encapsulated in the inside of a polyhedralprotein and a state where it is embedded while a part thereof is exposedto the outside of the polyhedral protein. In addition, examples of theshape of the complex include a regular shape such as cube, rectangularparallelepiped and cylinder, and an irregular shape such as aparticulate form. According to the shape, the amount of the encapsulatedtarget protein can be increased, the size of the target protein can beincreased, or a function such as a bioactive function or a catalyticfunction can be dramatically enhanced.

In the present invention, the restricted region of VP3 is a region fromthe 41st amino acid residue to the 79th amino acid residue as well as aregion from the N-terminus to the 40th amino acid residue. Incidentally,though it takes time and efforts and is inefficient, a region in which10 amino acid residues have been added to the N-terminus or theC-terminus of a region from the 41st amino acid residue from theN-terminus to the 79th amino acid residue can also be used.

Further, when considering the point of binding to a biologically relatedchemical substance, the target protein is an enzyme, an antigen, anantibody, a receptor or a cytokine, when considering the point of aphotochemical property, it is a light-emitting protein, and whenconsidering the point of an electron transfer reaction, it is ametal-binding protein or a metal ion-containing enzyme. When consideringthe point of such a property of a target protein, a constitution whichis selected from these and is at least one member is preferred.

A process for producing a protein complex according to the presentinvention comprises introducing a vector that has been integrated with aDNA encoding a target protein having a restricted region of VP3 as asignal simultaneously or together with a vector that has been integratedwith a DNA encoding a polyhedral protein into a cell such as an insectcell, an animal cell, a plant cell or an a cellular cell, and culturingthe cell under the conditions suitable for each cell. In this way, theprotein complex can be efficiently produced without lowering itsfunction. However, the vector that has been integrated with a DNAencoding a target protein and the vector that has been integrated with aDNA encoding a polyhedral protein are a plasmid vector, a virus vectoror the like, and it is only necessary to individually select the onesuitable for a cell into which a DNA is introduced.

As for use of a protein complex of the present invention, it can beapplied as a biosensor such as an immunosensor, a gene sensor or a lipidsensor by arranging and immobilizing the protein complex on a substrateto use the protein complex as a receptor, converting light amount ormass into an electrical signal by a transducer such as SPR, a photoncounter or a crystal oscillator, and displaying the electrical signal.As a material for the substrate, a glass, a plastic, a metal or the likecan be used. In addition, as a method of bonding the substrate and theprotein polyhedron, an adhesive such as gelatin or a macromolecularpolymer can be used.

In addition, by using a tubular container, in which a test solution canbe passed through, instead of the above-mentioned substrate, and packingthe protein complex in the container in such a manner that it can becontacted with a substance in the test solution, it can be applied as abiosensor.

Further, by preparing a particulate protein complex by the same methodas in Example 1 using a DNA encoding an enzyme such as a protease, alipase or an esterase having a catalytic ability, and packing theprotein complex in a container in any of various forms, it can beapplies as an immobilized enzyme having a catalytic ability.

The detail of the invention of this application will be described withreference to Examples. However, the invention of this application is byno means limited to these Examples.

EXAMPLE 1

The present invention will be described according to Examples and theattached figures to explain it in more detail.

(1) Preparation of a Virus Producing a Polyhedral Protein

In the case where a polyhedron is produced with IPLB-Sf21-AE (Sf21)derived from an insect cell Spondoptera frugiperda, to form a cubicpolyhedron of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), arecombinant virus (AcCP-H) that had been integrated with a polyhedralprotein gene of BmCPV strain H (Mori et al., (1993) J. Gen. Virol. 74,99-102) was inoculated. This AcCP-H is a recombinant virus that has beenintegrated with a polyhedral protein gene of strain H at the downstreamof the polyhedrin promoter of a baculovirus vector derived fromAutographa californica nucleopolyhedrovirus (AcNPV).

(2) Analysis of a Signal Composed of Only a Restricted Region of aCapsid Protein VP3

1) Shortening of BmCPV S4 Encoding a Capsid Protein VP3

A plasmid pVP3(XbaI)EGFP (International Patent Application WO02/36785A1) was digested with a restriction enzyme XbaI, and furtherdigested with a restriction enzyme KpnI. This DNA was dissolved in 100μl of ExoIII buffer in a tube, and 1 μl of Exonuclease III was added,stirred and incubated at 25° C. This DNA solution was sampled (5 μleach) at 30 second intervals and added to 100 μl of MB Nuclease Bufferwhich had been prepared in another tube. The mixture was incubated at65° C. for 5 minutes to inactivate Exonuclease III and cooled down to37° C. again. Then, 2 μl of Mung Bean Nuclease was added and the mixturewas incubated at 37° C. for 30 minutes. After performing phenolextraction and ethanol precipitation, DNA was dissolved in 50 μl ofKlenow Buffer, and 1 μl of Klenow Fragment was added. After the mixturewas incubated at 37° C. for 15 minutes, thereby completely repairing theends, 10 μl of the mixture was taken out and added to 100 μl of LigationSolution A which had been prepared in another tube. Further, 12 μl ofLigation Solution B was added and stirred, and the mixture was reactedat 16° C. for 3 hours. Then, ethanol precipitation and rise were carriedout. After the collected DNA was digested with a restriction enzyme XbaIfor 1 hour, the mixture was added to a competent cell JM109 (100 μl),whereby transformation was carried out. Incidentally, theabove-mentioned procedure was carried out by using, for example,Kilo-Sequence Deletion Kit (manufactured by Takara Co.) according to itsprotocol (FIG. 1).

2) Construction of a Recombinant Transfer Vector

The transformed E. coli was plated on a 2×TY plate containing kanamycinand cultured overnight at 37° C. The formed colonies were culturedovernight at 37° C. in 2×TY medium containing kanamycin. The plasmid DNAwas extracted, digested with restriction enzymes BglII and BamHI andelectrophoresed. It was confirmed that the DNA fragment was shortened,and a sequence analysis was performed, whereby the nucleotide sequenceof the DNA fragment was confirmed. The plasmid DNA solution which wasrequired for confirmation of the nucleotide sequence was digested with arestriction enzyme NotI, and inserted at the NotI site of a baculovirustransfer vector pVL1392 (manufactured by PHARMINGEN). A competent cellJM109 (100 μl) was transformed with this vector, plated on a 2×TY platecontaining ampicillin and cultured overnight at 37° C. The formedcolonies were cultured overnight at 37° C. in 2×TY medium containingampicillin. The plasmid DNA was extracted, and a sequence analysis wasperformed. From the results of the analysis, the one in which the insertwas inserted in the right direction was selected, which was used as arecombinant transfer vector pAcVP3(x)/EGFP (with the proviso that xrepresents the number of bases of the S4 cDNA encoding VP3 of BmCPV)(FIG. 2).

3) Construction of a Recombinant Baculovirus

A cultured insect cell Sf21 was cotransfected with the constructedrecombinant transfer vector pAcVP3 (x)/EGFP (5 μg each) and a linearBaculogold Baculovirus DNA (0.5 μg) (manufactured by PHARMINGEN)according to the lipofectin method. Subsequently, the plaque waspurified, whereby a recombinant virus AcVP3(x)/EGFP was constructed.

(3) Preparation of a Protein Complex Containing EGFP as a Target Protein

1) Expression of the Recombinant Protein in Sf21 Cell

As a control, double infection with AcVP3/GFP (Ikeda et al., (2001) J.Virol. 75, 988-995) and AcCP-H (Mori et al., (1993) J. Gen. Virol. 74,99-102) or with AcVP3(XbaI)/GFP (International Application WO02/36785A1) and AcCP-H, was performed. On the other hand, for thepurpose of shortening VP3, double infection with AcVP3(x)/GFP and AcCP-Hwas performed. The double infection was performed at 10 p.f.u./cell foreach case. After the virus was allowed to adsorb to cells at roomtemperature for 1 hour, the virus solution was removed, and 2 ml ofTC-100 containing 10% fetal bovine calf serum was added, and the mixturewas incubated at 27° C. for 4 days.

2) Purification of Polyhedra

The cubic polyhedra were collected from the infected cells on the 4thday. After washing with PBS (20 mM NaH2PO4, 20 mM Na2HPO4, 150 mM NaCl,pH7.2), the polyhedra were homogenized in ice with a homogenizer. Thehomogenate was washed with 1% Tween 20, and the polyhedra were collectedby centrifugation. Then, centrifugation with the sucrose densitygradient from 1.5 M to 2.2 M at 50,000×g for 45 minutes was performed toextract the fraction of polyhedra. The extracted sample was washed withPBS, followed by centrifugation at 15,000×g for 10 minutes, and purifiedpolyhedra were collected.

3) Determination of Encapsulation of EGFP in a Polyhedron

Polyhedra from cells subjected to double infection with AcVP3(X)/GFP andAcCP-H, and as a control, AcVP3/GFP and AcCP-H, and AcVP3(XbaI)/GFP andAcCP-H were purified, and encapsulation of EGFP in a polyhedron wasdetermined based on the presence or absence of fluorescence from thepolyhedron using a fluorescence microscope (manufactured byOLYMPUS-IX71) (FIG. 3). As a result, in any case, green fluorescencefrom the polyhedron was observed, and it was confirmed that VP3/GFP orVP3(XbaI)/GFP was encapsulated in the polyhedron.

Subsequently, for all AcVP3 (X)/GFP prepared as shown in FIG. 2,encapsulation of EGFP in the polyhedron was investigated. As a result,it was found that a VP3(250)/GFP molecule encoded by a chimeric gene inwhich a region containing from the 5′-terminus to the 250th base of theVP3 gene had been introduced into the 5′-terminus of the EGFP gene wasembedded in the polyhedron. That is, it means that a signal forembedding a protein molecule specifically in the polyhedron (embeddingsignal for polyhedron) exists in a region up to the 79th amino acidresidue at the N-terminus of VP3. Because of the existence of thissignal, a VP3(250)/GFP molecule is encapsulated in the polyhedron, andas a result, green fluorescence from the polyhedron could be observed asshown in FIG. 3.

However, in the case of a chimeric gene in which a region containingfrom the 5′-terminus to the 130th base of the VP3 gene had beenintroduced into the 5′-terminus of the EGFP gene, a fusion GFP moleculeVP3(130)/GFP encoded by this chimeric gene lost the function of beingencapsulated in the polyhedron, and green fluorescence from thepolyhedron was not observed at all (FIG. 3). This indicates that theembedding signal for polyhedron does not exist in the region up to the39th amino acid residue at the N-terminus of VP3.

Further, a fragment from the 135th base to the 292nd base of VP3 wasamplified by the PCR method, and a chimeric gene in which the amplifiedfragment was introduced into the 5′-terminus of the EGFP gene wasconstructed. As a result, a region encoding from the 41st amino acidresidue at the N-terminus to the 93rd amino acid residue of VP3 isintroduced into the N-terminus of EGFP. It was confirmed whether thisVP3(135-292)/EGFP was encapsulated in the polyhedron in a similarmanner, as a result, green fluorescence from the polyhedron wasobserved.

From the above result, for embedding of a protein molecule in apolyhedron via VP3, a very limited N-terminal region of VP3, that is, aregion from the 41st amino acid residue at the N-terminus to the 79thamino acid residue of VP3 is found to function as an embedding signalfor polyhedron.

Effect of Shortening of VP3

By introducing a gene encoding a region with different length from the5′-terminus of the VP3 gene into the 5′-terminus of the GFP gene,regions of various amino acid sequences derived from VP3 were introducedinto the N-terminus of GFP. The color development of green fluorescenceby a fusion GFP molecule expressed from any of these chimeric genes wascompared. As a result, as shown in FIG. 4, as the region of VP3 to beintroduced into the N-terminus of GFP became shorter, the colordevelopment of green fluorescence was increased. However, in the casewhere the region was made shorter than the 79th amino acid residue fromthe N-terminus of VP3, the color development of green fluorescence wassubstantially the same. In this way, in the case where another aminoacid sequence is introduced into a target protein, as the length of thesequence becomes shorter, the bioactivity of the target protein isincreased. However, the sequence becomes shorter than necessary, thefunction as the signal will be lost.

The signal for encapsulating a target protein in the polyhedron of VP3obtained in the present invention has a function sufficient forencapsulating the target protein in the polyhedron when it wasintroduced in the target protein molecule. Moreover, the signal has alength that does not disturb the bioactivity of the target protein.Further, it is indicated that by shortening the length of VP3 accordingto the present invention, a molecule which is larger by the length ofVP3 that had been removed can be embedded in the polyhedron, therefore,the effect of the present invention is high.

Subsequently, according to the procedure of Example 1, a biosensor usinga cubic protein complex about 10 μm on a side by applying human-derivedCyclin-dependent kinase (CDK5) as a target protein will be explained.

EXAMPLE 2

A biosensor was prepared by arranging a complex on a slide glass.

On a slide glass, 5 μl of a gelatin solution (gelatin: 0.5, Crk: 0.02)was dropped. Incidentally, Crk is chromium potassium sulfate (anantiseptic).

The front sides of the slide glass and a new slide glass were puttogether carefully. When the solution was spread therebetween, the slideglasses were slowly pulled apart. After the gelatin was completelydried, 1 μl of a complex solution which had been well stirred wasdropped thereon, then dried, whereby a biosensor was prepared. Thissensor was immersed in distilled water until use.

Incidentally, a complex solution represents a solution obtained bypurifying a complex which has been expressed in a large amount in Sf21cells, and suspending the purified complex in distilled water.

Verification

Verification method

(1) Suppression of Peroxidase Activity

A hydrogen peroxide solution (adjusted to a final concentration of 1% byPBS) was placed on the part where the complex was dropped. After a15-minute treatment at room temperature, washing was carried out withPBS (in order to remove the hydrogen peroxide solution).

In this way, the peroxidase activity to be a background was suppressed.

(2) Blocking with Normal Serum (5% NHS)

Normal horse serum was adjusted to a final concentration of 5% with PBScontaining 0.3% Triton X-100 (T-PBS), and added to the slide glass.After a 20-minute treatment at room temperature, washing was carried outwith T-PBS.

(3) Primary Antibody Reaction

An anti-Cdk5 monoclonal antibody was diluted to 100-fold with T-PBScontaining 5% serum, and reaction was carried out at 37° C. for 3 hours.Then, washing was carried out with T-PBS.

(4) Biotinylated Anti-Mouse IgG Antibody Reaction

A biotinylated secondary antibody was diluted to 100-fold with T-PBS,and reaction was carried out at 37° C. for 1 hour. Then, washing wascarried out with T-PBS.

On the other hand, A solution and B solution to be used in ABC reactionwere diluted to 100-fold with T-PBS, and reaction was carried out for atleast 30 minutes in advance.

(5) Reaction with ABC Reagent (VECTASTAIN ABC KIT STANDARD PK-6100)

After 1-hour reaction at room temperature, washing was carried out withT-PBS.

(6) Washing

Since precipitate is formed by the reaction of the subsequent DAB withphosphoric acid, in order to replace PBS, washing was carried outlightly with 50 mM Tris-HC (pH 7.5), and the solution was replaced.

(7) Incubation with DAB Substrate

DAB powder was added to 50 mM Tris-HCI solution at a concentration of 50mg/ml, 16 μl of hydrogen peroxide solution was further added, andreaction was carried out at room temperature for 25 minutes. After thereaction, the slide glass was immersed in 50 mM Tris-HCI solution.

(8) Encapsulation with Glycerol/PBS

After the slide glass was dried, one drop of glycerol/PBS was dropped onthe sample, then a cover glass was placed thereon avoiding any airbubble under.

By using the above-mentioned verification method, an antigen-antibodyreaction was attempted with a protein complex having a target proteinencapsulated therein and a polyhedron without any protein encapsulatedtherein. The results are shown in FIG. 5. As shown in FIG. 5, as for theprotein complex having Cdk5 encapsulated therein, the antigen-antibodyreaction of the Cdk5 molecule and the anti-Cdk5 antibody could beobserved on its surface. In this way, an antigen-antibody reaction, thatis, a protein-to-protein interaction between an antigen protein and anantibody protein can be observed on the surface of a protein complexhaving a fused target protein encapsulated therein.

INDUSTRIAL APPLICABILITY

As described in detail above, according to the present invention, aprotein complex comprising a polyhedral protein and a target protein canbe efficiently produced by introducing a polyhedral protein, which is aconstituent protein of a polyhedron having essentially an insect virusencapsulated therein and only a restricted region of a capsid proteinVP3 of cytoplasmic polyhedrosis virus as a signal into a target protein.

In addition, a protein complex obtained by encapsulating a proteinmolecule having a bioactive function such as an enzymatic activity, anantigen-antibody reaction or a protein-to-protein interaction in apolyhedral protein can be used as an excellent biosensor or immobilizedenzyme.

1. An isolated protein complex comprising: a polyhedral protein ofBombyx mori strain H cytoplasmic polyhedrosis virus (CPV) having Bombyxmori strain H CPV encapsulated therein; and a target protein directlyfused to the C-terminus of a fragment of a capsid protein VP3 of Bombyxmori strain H CPV, wherein said fragment consists of the 41st to 79thamino acid residues of SEQ ID NO: 2 and is embedded in the polyhedralprotein, and wherein said target protein is heterologous with respect tosaid fragment and is encapsulated by the polyhedral protein.
 2. Theisolated protein complex according to claim 1, wherein the polyhedralprotein has an effect on improvement in the stability of the targetprotein, protection thereof or improvement in the preservation propertythereof, or a combination of any of these.
 3. The isolated proteincomplex according to claim 1, wherein the target protein is at least onemember selected from the group consisting of fluorescent orlight-emitting proteins, enzymes, antigens, antibodies, cytokines,receptors and bioactive proteins.
 4. A process for producing an isolatedprotein complex, comprising the steps of: infecting a cell with a vectorthat has been integrated with a nucleic acid encoding a fragment of acapsid protein VP3 of Bombyx mori strain H cytoplasmic polyhedrosisvirus (CPV) directly fused to a nucleic acid encoding a target proteintogether with a recombinant virus that has been integrated with a geneencoding a polyhedral protein of Bombyx mori strain H CPV, and culturingthe infected cell, whereby a protein complex comprising a polyhedralprotein of Bombyx mori strain H CPV having Bombyx mori strain H CPVencapsulated therein and a target protein directly fused to theC-terminus of a fragment of a capsid protein VP3 of Bombyx mori strain HCPV is produced in the cell, wherein the fragment of the producedprotein complex consists of the 41st to 79th amino acid residues of SEQID NO: 2 and is embedded in the polyhedral protein, and wherein saidtarget protein is heterologous with respect to said fragment and isencapsulated by the polyhedral protein.
 5. A biosensor comprising: anisolated protein complex comprising: a polyhedral protein of Bombyx moristrain H cytoplasmic polyhedrosis virus (CPV) having Bombyx mori strainH CPV encapsulated therein; and a target protein directly fused to theC-terminus of a fragment of a capsid protein VP3 of Bombyx mori strain HCPV, wherein said isolated protein complex is arranged in dots or lineson a substrate and immobilized thereon, wherein the fragment consists ofthe 41st to 79th amino acid residues of SEQ ID NO: 2 and is embedded inthe polyhedral protein, and wherein said target protein is heterologouswith respect to said fragment and is encapsulated by the polyhedralprotein.
 6. A biosensor according to claim 5, wherein said isolatedprotein complex is packed in such a manner that said isolated proteincomplex is to be contacted with a substance in a test solution in arecess formed on a substrate.
 7. A biosensor according to claim 5,wherein said isolated protein complex is packed in a container in such amanner that said isolated protein complex is to be contacted with asubstance in a test solution.
 8. An isolated protein complex comprising:a polyhedral protein of Bombyx mori strain H cytoplasmic polyhedrosisvirus (CPV) having Bombyx mori strain H CPV encapsulated therein; and atarget protein directly fused to the C-terminus of a fragment of acapsid protein VP3 of Bombyx mori strain H CPV, wherein said targetprotein is an enzyme, is heterologous with respect to said fragment, andis encapsulated by the polyhedral protein, and wherein said fragmentconsists of the 41st to 79th amino acid residues of SEQ ID NO: 2 and isembedded in the polyhedral protein.